1. Field of the Invention
The present invention pertains to the art of electrolytic sensing systems, and more specifically, to an apparatus and method for sensing a time varying ionic current in an electrolytic environment having improved sensitivity and bandwidth.
2. Discussion of the Prior Art
The measurement of the electrical conductivity of small fluid regions is of importance in many applications. For example, small particles, such as viruses, suspended in a fluid can be counted using a resistive pulse technique, i.e. measuring the momentary change they produce in the average fluid conductance as they pass through an orifice. In recent years, it has become of great scientific and technological importance to measure the variation in electrical conductivity of entities that span the membrane wall of a cell. Such entities, which include protein pores, ion channels, transporters, and related entities (herein denoted by the group name: ion channels), control the passage of specific ions into and/or out of a cell. Measuring the ion fluxes by the electrical current they carry is much easier, faster, and more fundamental than by other methods such as a radioactive tracer.
Electrical measurements of ion channel activity go back to Hodgkin and Huxley's identification of currents flowing through homogeneous ensembles of channels that they called “ionic conductances”. Measurements of currents through single channel proteins, one molecule at a time, began with the work of Haydon and Hladky. They found that currents through single gramicidin channels had a rectangular time course, and that the duration of opening corresponded to the time-dependent processes studied by Hodgkin and Huxley, while the amplitude of the current through the channel corresponded to the “instantaneous current” recorded by them.
Hundreds of types of ion channels have been investigated to date with electrical methods, leaving thousands of ion channels and transporters yet to study. Channel currents vary from the currently unmeasurable (˜0.2 Pico Amps (pA)) to hundreds of pA. Indeed, many of the biologically and medically important ion channels have unmeasurable single channel currents, and their conductance must be estimated from macroscopic measurements of many ion channels. In addition to a need to be able to record smaller current changes, it is generally believed that the speed of many ion channel transitions is faster than the response time of existing recording systems and thus these channels have gone unmeasured.
The principal advance in recording ion channels was made by Sakmann and Neher who developed a patch pipette method that allowed recording of single channel currents with a thin-walled, drawn-glass pipette pressed against a cell membrane. One example of such a system is depicted in FIG. 1. Although not shown, the entire patch clamp apparatus would typically be enclosed in an electrically conducting shield to minimize the pick-up of electromagnetic noise. It was found that the measurement fidelity could be increased significantly by drawing the membrane several microns into the pipette by suction so as to increase the area of the membrane in contact with the surface of the glass. The norm is for the pipette to be positioned by hand. In automated cell patching systems, cells are drawn by suction to a micron-scale orifice in a glass or silicon surface. Regardless, once the cell is attached, an electrical circuit across an ion channel must be completed by penetrating the cell membrane with a sharp electrode or tearing off a patch of cell membrane attached to the measurement region to allow the part of the ion channel on the inside surface of the cell to be exposed to the electrolyte in the bath.
An alternate, more direct, route is to create a membrane incorporating an ion channel and either suspend the membrane over an orifice (typically in the order of 100 microns or micrometers (μm) in diameter) in a solid material such as Teflon, or support it with a solid electrode surface, possibly via polymer tethers. One example of such a suspended artificial membrane system is depicted in FIG. 2. Although not shown, this system would typically be enclosed by an electrically conducting shield. The former method permits macroscopic access from both sides of the membrane, but is very fragile. The latter is robust, but the chemistry of the lipid and polymer tethers requires that an inert electrode (e.g. gold) that has a predominantly capacitive (i.e. alternating current (AC)) coupling to the electrolyte must be used. In addition, the very small fluid volume in the region between the membrane and solid surface prevents the use of traditional direct current (DC) methods to measure channel current because of the build up of ionic concentration gradients across the membrane.
Regarding measurement sensitivity and bandwidth, conventional ion channel recordings are limited by the current noise of the first stage amplifier, and the effective current noise produced by the voltage noise of the first stage amplifier acting on the total capacitance at the amplifier input.
Referring to FIG. 1, a typical patch clamp system 200 including a membrane 201 and an electrolyte bath 202 has the following properties. The input capacitance of a first stage amplifier 204 is 15 picofarads (pF). The capacitance of a pipette holder 208 varies from 1-5 pF, and the capacitance of a pipette 212 is 1-5 pF also. Larger pipette capacitances are associated with holders that have metallic shielding (Axon Instruments). However, the immersion of the thin walled tip of the patch pipette into the bath 202 produces a further capacitance in the order of 1 pF/mm. Although it is possible to immerse a pipette tip by only a very small distance, it is cumbersome to set up, and difficult to implement for any length of time because of the fluid level change due to evaporation. For a typical tip immersion distance of 5 millimeters (mm), the extra capacitance is 5 pF, and is a fundamental result of the pipette tip having a very thin wall. The capacitance of a wire 220 running from an electrode 224 to the system electronics depends on the wire diameter and length. Assuming the wire is 1 mm in diameter and 20 centimeters (cm) long, and the shield (not shown) is from 10 cm to 1 meter (cm) away, the wire capacitance is 1.5-2.1 pF. Taken together, the total capacitance at the amplifier input is the range of 25-30 pF.
The capacitance of the artificial membrane apparatus 228 is typically higher. Referring to FIG. 2, the same capacitance values are found for a first stage amplifier 230 and connecting wires 234 that connect first stage amplifier 230 to electrodes 236, but a membrane 238 suspended on a Teflon sheet 239 that divides two fluid volumes 240 and 242 has an area 1,000-10,000 times larger than membrane 201 of the patch pipette system. This produces an additional shunt capacitance in the range of 5-100 pF. In addition, the capacitance of the measurement side of the electrolyte bath or fluid volume 240 produces a capacitance that scales linearly with the bath radius and is typically at least 1 pF. Overall, the total capacitance at the amplifier input of the prior art artificial membrane apparatus is 22-120 pF.
The above prior art systems are well optimized in the sense that the internal contributions to the measurement noise are approximately equal. For example, the current noise and the voltage noise acting on the total input capacitance are approximately equal. However, this represents a trade-off in the first stage amplifier design for the known input impedance, and the two conventional types of measurement apparatus ultimately reach limitations inherent to their mechanical designs. For the patch pipette, the limitation is the pipette itself. For the Teflon cup, the limitation is the area of the membrane. As a result, in the twenty or so years that patch pipette and the related artificial bilayer measurements have been performed, there have been only marginal sensitivity and bandwidth improvements, and these have been almost entirely due to advances in electronics technology.
Based on the above, there exists a need for a system that will make a substantial improvement in ion channel recordings. Such a system must provide the required electrical properties to improve upon the sensitivity and bandwidth obtained by the well-optimized existing technology, and must also be no more difficult to use, and no more expensive than current technology.